The present invention relates to an improved method of windowing eggs, particularly fertilized eggs. The present invention further relates to methods for the resealing of eggs using a heat softened composition.
The wide interest in manipulating the avian genome, for applications ranging from enhancing traits of domestic poultry to the production of heterologous proteins within the egg, has resulted in intensive efforts to apply transgenic technology to poultry. In developing strategies for gene transfer, several features are desired including the ease of obtaining large numbers of embryos for manipulation, ease of accessing the embryo, and a low level of mosaicism. However, gaining access to the early embryo has posed several challenges due to the complex early life history of the avian embryo.
Access to the early embryo without jeopardizing future embryo development is critical to successful in vivo gene manipulation in birds. When laid, the avian embryo consists of a blastoderm containing 30,000-60,000 cells on top of the yolk and encased in a hard calcified egg shell. Immediately below the shell is the egg shell membrane that surrounds the egg white, the egg yolk and the developing embryo. The hard egg shell and large yolky egg of the avian embryo pose a significant obstacle to manipulating the embryo.
The first 24 hours of development of the chicken embryo take place within the oviduct of the hen, producing an embryo that is packaged for morphogenesis and growth over the next 21 days (Romanoff and Romanoff, 1949). Many procedures, including transgenic modification of the avian genome, require access to the interior of the egg. For example, in one method of modifying the genetic material of a chicken, a small volume of liquid containing retroviral transducing particles or transfected donor cells must be injected into the subgerminal cavity of the recipient embryo. In addition, it may be desirable to expose the developing embryo to antigens, viruses, vaccines, or growth factors. Disrupting the environment of the embryo during development can adversely affect its chance of survival (eg. Mann et al., 1973). Despite these challenges, several strategies have been devised to make gene transfer possible in poultry. Each targets a different stage of embryonic development, and each has varying degrees of success.
One approach targets the pluripotent Stage X blastoderm (Eyal-Giladi and Kochav, 1976) from a freshly oviposited egg (Eyal-Giladi, 1984). In one method to access the embryo, a small window is drilled through the egg shell and genetically modified cells from a donor Stage X embryo (Eyal-Giladi and Kochav, 1976) or retroviral particles are injected into the subgerminal cavity immediately below the blastoderm (Bosselman et al., 1989; Brazolot et al., 1991; Fraser et al., 1993; Thoraval et al., 1995). To provide access to the interior of the egg and the embryo, the underlying egg shell membrane is cut away with a scalpel and 2-10 microliters of experimental solution is microinjected into the embryo. The hole is then sealed in one of several ways.
Most commonly, the hole is covered with fresh egg shell membrane from a donor egg, with the membrane applied in the same orientation as in the egg, i.e., albumen-side down. When the membrane dries, it is permanently sealed with plastic model cement or a gas permeable surgical membrane. See also Carsience et al. (1993), and Fraser et al. (1993). Other similar methods have been used to access the developing embryo. Thus, Thoraval et al. (1995) remove a triangular piece of shell, inject 10 xcexcl of experimental solution through the opening into the embryo, then seal the egg by replacing the shell piece and covering it with adhesive tape. Marzullo, G. (1970) cuts a hole in the shell, covers it with a glass cover slip, and seals it with paraffin wax. This procedure is fast and easy, allowing large numbers of embryos to be manipulated. However, this approach results in compromised embryo survival. Typically, less than 10% hatch (Petitte et al., 1990; Thoraval et al., 1994, 1995). It is believed that residual air within the egg after sealing the window results in embryonic defects and death (Mann et al., 1973; Fisher and Schoenwolf 1983; Fineman et al., 1986; Fineman and Schoenwolf 1987).
Direct comparison of the various published results is complicated, however, because the time of windowing and termination varied. Fineman et al. (1986) found that survival rates increased as the incubation time prior to windowing increases. In addition, the percentage of abnormal embryos observed using this windowing method increased as the incubation time after windowing increased (Fisher and Schoenwolf 1983; Fineman and Schoenwolf 1987).
Culturing Stage X embryos (Eyal-Giladi and Kochav, 1976) through a series of surrogate shells (Perry, 1988; Naito and Perry, 1989; Naito et al., 1990) improves hatch rates to between 20% and 60%. However, this method is labor-intensive and time-consuming, and defeats the advantage of manipulating this stage of development.
Speksnijder et al, in U.S. Pat. No. 5,897,998, teach filling the air space created during windowing with an aqueous solution before sealing to increase hatchability of eggs subject to manipulation. However, the present inventors found that, in as many as 40% of the eggs, the method of Speksnijder et al results in the presence of air bubbles immediately below the air sac at candling, resulting in decreased hatchability. Candling the eggs after windowing revealed that these bubbles were not present prior to incubation, suggesting that an imperfect seal may be formed using the method of Speksnijder et al.
What is needed, therefore, is a method of windowing avian eggs that avoids the pitfall of early death of the embryo, thereby making windowing effective for the rapid and efficient production of transgenic birds. What is further needed is a method to minimize residual air in the egg during the windowing process before sealing the egg.
These and other objectives and advantages of the invention will become fully apparent from the description and claims that follow or may be learned by the practice of the invention.
This invention provides a method for manipulating the contents of eggs. A first opening is made in the egg shell, and the underlying egg shell membrane is cut away. After manipulation of the egg or embryo, the opening in the egg shell is resealed by applying a heat softened composition, such as a hot melt glue. Any air bubbles introduced into the interior of the egg upon cutting of the underlying egg shell membrane can optionally be removed by expanding the air sac of the egg through a second opening in the egg shell and forcing undesirable air bubbles out through the first opening by displacement of the egg white and the yolk. The egg is manipulated as described above and then incubated to allow development of the embryo. The incubation is maintained until the embryo is hatched from the egg. The invention improves hatchability of fertilized eggs following embryo manipulation.
Suitable eggs for application of the methods of the present invention are avian eggs including, but not limited to, eggs of the ratite, chicken, turkey, quail, duck, pheasant and goose. The egg may contain an embryo. The invention is suitable for any commercial application requiring accessing an egg and microinjection of a solution into the egg and/or an embryo. For example, genetically modified cells, attenuated viruses, antigens, growth factors and cytokines may be microinjected. The invention provides improved hatchability following the incubation of manipulated fertilized eggs. This aspect may be useful when large numbers of injections are required, for example, in the production of genetically manipulated and transgenic animals.
Additional objects and aspects of the present invention will become more apparent upon review of the detailed description set forth below when taken in conjunction with the accompanying figures, which are briefly described as follows.